“Feature-level” analysis of microarray data identifies a role for Aire in releasing paused Pol-II. (A) Expression values for individual microarray features were analyzed relative to whole-transcript averages, and “imbalanced” exons in datasets from MECs from Aire-KO and Aire-WT mice were flagged (PLATA t test P < 0.05). The proportion of imbalanced exons is shown for 420 transcripts most strongly induced by Aire, or an equal number of expression-matched Aire-neutral genes, or 84 transcripts repressed by Aire (Table S1). (B) Exon-level expression was normalized to the whole-transcript average in either WT or Aire KO datasets. Transcripts with imbalanced exon1 representation are shown in red (Left) or imbalanced representation of internal exons in purple (Right). (C) The expression level of each feature for transcripts showing an exon1 imbalance was plotted relative to its distance from the TSS for Aire-induced genes, in Aire KO or WT MECs. (D) Expression level of each feature for all Aire-induced (Left; overall WT/KO ratio >3) or Aire-neutral genes (Right) plotted as a function of distance from the TSS, either individually (Upper) or grouped in 25-bp bins (Lower). (E) The expression level of each feature for transcripts showing an exon1 imbalance was plotted relative to its distance from the TSS for Klf4-induced genes, in Klf4 KO or WT cells (data from ref. 35). A “TSS proximal–distal” index was calculated in the KO samples as the ratio of the medians of expression values in proximal (<200 bp from TSS) vs. distal features.

Validation of the microarray-based detection of stalled polymerases. (A) RT-PCR analysis of the effect of flavopiridol on AIRE-transfected 4D6 cells for a panel of AIRE-sensitive or -insensitive genes. Relative expression levels are shown as the mean ± SD from three replicates. (B) 4D6 epithelial cells were treated with flavopiridol for 24 h before expression profiling. The expression level of each microarray feature for flavopiridol sensitive transcripts showing an exon1 imbalance is plotted vs. its distance from the TSS, in flavopiridol-treated and control 4D6 cells. (C) A “TSS proximal–distal” index was calculated in KO samples for several transcription factors from plots such as those of Fig. 1C as the ratio of the medians of expression values in proximal (<200 bp from TSS) vs. distal features. TSS “proximal–distal” indices calculated for sensitive genes, in expression datasets of KO of factors involved in the release of stalled Pol-II (black) or of classic transcription factors impacting initiation (gray).

Aire accumulates at the TSS of Pol-II–rich genes. (A) ChIP-seq analysis was performed in control or Aire-transfected 293 cells. Binding of Aire (Upper) and total Pol-II (Lower) over the whole length of an Aire-induced (THAP10) and an Aire-neutral gene (ZBTB25), shown as examples. (B) As in A, close-up representation of the TSS region of an Aire-induced (INTS12) and an Aire-neutral gene (NME1). (C) Cumulative distribution of Aire and Pol-II in a 4-kb window around the TSS of 2,272 genes. (D) Distribution of total Pol-II and Aire in 25-bp bins in a window of 4 kb around the TSS of all genes, ranked by expression values. (E) As in D, representation of Aire-bound DNA in an independent experiment at two different times after Aire transfection; Right: chromatin precipitated with control IgG (normal mouse IgG1).

Aire activates transcripts with a surfeit of stalled polymerases. (A) Mean signal density of Pol-II in windows (−300 to +200 bp) around the TSS plotted against the microarray expression levels of the corresponding gene. Gray, all Aire-neutral genes; red, Aire-induced genes; blue, Aire-repressed genes. Red, blue, and green lines are spline fits for Aire-induced, Aire-repressed, and neutral genes, respectively. (B) Mean density of total Pol-II in 25-bp bins around the TSS of Aire-induced and Aire-neutral genes. (C) Effect of Aire transfection: as in A, mean Pol-II signal density around the TSS plotted against microarray expression levels, in control (Left) or Aire-transfected (Right) 293 cells. Color coding as in A.